Humans with point mutations in MYH9, the gene encoding nonmuscle myosin heavy chain (NMHC) IIA, develop a variety of syndromes including defects in their platelets (macrothrombocytopenia), kidneys (glomerulonephritis) and granulocytes (inclusion bodies). More than 30 different mutations in MYH9 have been reported to date, including both missense and nonsense mutations. The purpose of these studies is to gain insight into the pathological mechanism of the diseases caused by these mutations by creating mouse models for three of the mutations (R702C in motor domain;D1424N and E1841K in rod domain) and studying the resultant mouse phenotypes. Previous in vitro work has shown that the R702C mutation, which is in the motor domain of NMHC IIA compromises the MgATPase activity and is responsible for the movement velocity of the myosin;while mutations D1424N and E1841K in the rod domain may affect NMHC IIA filament formation. We have produced both R702C and D1424N mutant mice by using homologous recombination to replace wild type NMHC IIA with mutant R702C or D1424N in NMHC IIA. The E1841K mutant mice were obtained from Dr. M Kelley at Duke University. Heterozygous D1424N and E1841K mutant mice produce homozygous mutant offspring at close to normal ratios. By contrast, homozygous Arg702Cys mice die at embryonic day (E)10.5 to 11.5, which is considerably later in development than knockout myosin II-A mice (E6.5). These results indicate that the motor domain function of NMHC IIA is critically important during embryonic development. Interestingly, giant platelets accompanied by a mild decrease in platelet count are found in the blood smears from all adult heterozygous mice with the point mutations. The homozygous D1424N and E1841K adult mice have even larger platelets than the heterozygotes which are accompanied by abnormally low platelet counts. Results of bone marrow histology study are consistent with failure of platelet release into the circulation. Heterozygous mutant mice also display prolonged bleeding times. Some, but not all adult heterozygotes from all mutant lines have higher urine albumin/creatinine ratios at 8-9 weeks, suggesting that glomerular disease is developing in these mice. Light and transmission electron microscopy studies show focal segmental glomerulosclerosis in the heterozygous R702C and both the heterozygous and homozygous D1424N and E1841K mice, while the basement membranes appear normal. Our results show that even heterozygous mutations in the mouse Myh9 gene can reproduce human MYH9-related diseases. These mouse models will be useful in understanding the pathophysiology of human MYH9-related diseases and should also be useful in designing and developing therapies. In addition to using these mutant mice to study the relation between the nonmuscle myosin II-A mutation and disease, we plan to use various cells derived from these mice to study the effects of the mutation on basic properties of the cell. These include cell-cell and cell matrix adhesion, cell polarity and cell migration. To gain clear insights into the distribution and function of different isoforms of nonmuscle myosin II (NMII) in normal mice, the enhanced GFP or mCherry sequence has been inserted in front of the start codon of the Myh9 gene in the first coding exon. We have obtained both heterozygous and homozygous GFP or mCherry tagged NMIIA mice. The expression level of the tagged NMIIA is similar to that of the endogenously expressed untagged NMIIA in both heterozygous mutants. This tagged NMIIA mouse model will shed light on the function of NM IIA in development. Various cell lines derived from the mice will be used to study the regulation and function of NM IIA in adhesion, cell polarity and cell migration. We also plan to cross mCherry tagged NM IIA mice with GFP tagged NMIIB mouse. The offspring with mCherry tagged NMIIA and GFP tagged NMIIB should be useful to study if NMIIA and NMIIB can form copolymers in vivo. The cell lines (e.g. fibroblasts) derived from this mouse can be used to study the various biological properties of NMIIA and NMIIB in cell polarity and migration with confocal or TIRF microscopy. The purpose of an additional study is to learn whether one isoform of NM II, specifically NM IIC, can functionally replace a second one, NM IIA, in mice. To replace NM IIA with NM IIC, homologous recombination wasl be used to inactivate NM IIA by inserting the cDNA for NM IIC-GFP into the first coding exon of the Myh9 gene. We have obtained heterozygous NM IIC replacing NM IIA mice. However, breeding of heterozygous mutant mice does not produce homozygous NM IIC replacing NM IIA mice. We plan to investigate at which embryonic stage the homozygous mice die and the impact of NM IIC replacing NM IIA on cell polarization and migration.